High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion

Rabphilin is a protein that associates with the GTP-bound form of Rab3, a s mall GTPase that controls a late step in Ca2+-triggered exocytosis, Rabphil in is found only in neuroendocrine cells where it co-localises with Rab3A o n the secretory vesicle membrane, The Rab3 binding domain (residues 45 to 1 70), located in the N-terminal part of Rabphilin, includes a cysteine-rich region with two zinc finger motifs that are required for efficient interact ion with the small GTPase, To determine whether binding to Rab3A is necessa ry for the subcellular localisation of Rabphilin, we synthesised point muta nts within the Rab3-binding domain. We found that two unique mutations (V61 A and L83A) within an amphipathic a-helix of this region abolish detectable binding to endogenous Rab3, but only partially impair the targetting of th e protein to secretory vesicles in PC12 and pancreatic HIT-T15 cells. Furth ermore, both mutants transfected in the HIT-TIS beta cell line stimulate Ca 2+-regulated exocytosis to the same extent as wild-type Rabphilin, Surprisi ngly, another Rabphilin mutant, R60A, which possesses a wild-type affinity for Rab3, and targets efficiently to membranes, does not potentiate regulat ed secretion. High affinity binding to Rab3 is therefore dispensable for the targetting o f Rabphilin to secretory vesicles and for the potentiation of Ca2+-regulate d secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.